Respiratory syncytial virus (rsv) vaccine

Abstract

The present invention relates to an mRNA sequence, comprising a coding region, encoding at least one antigenic peptide or protein of RSV infections Respiratory syncytial virus (RSV) or a fragment, variant or derivative thereof. Additionally the present invention relates to a composition comprising a plurality of mRNA sequences comprising a coding region, encoding at least one antigenic peptide or protein of RSV infections Respiratory syncytial virus (RSV) or a fragment, variant or derivative thereof. Furthermore it also discloses the use of the mRNA sequence or the composition comprising a plurality of mRNA sequences for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the prophylaxis or treatment of RSV infections Respiratory syncytial virus (RSV) infections. The present invention further describes a method of treatment or prophylaxis of RSV infections using the mRNA sequence.

1 . mRNA sequence comprising a coding region, encoding at least one antigenic peptide or protein derived from the fusion protein F, the glycoprotein G, the short hydrophobic protein SH, the matrix protein M, the nucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2 protein, the phosphoprotein P, the non-structural protein NS1 or the non-structural protein NS2 of Respiratory syncytial virus (RSV), or a fragment, variant or derivative thereof;
 wherein the G/C content of the coding region is increased compared with the G/C content of the coding region of the wild type mRNA, and wherein the coded amino acid sequence of said GC-enriched mRNA is preferably not being modified compared with the coded amino acid sequence of the wild type mRNA.   
     
     
         2 . The mRNA sequence according to  claim 1 , wherein the coding region encodes the full-length protein of fusion protein F, nucleoprotein N or glycoprotein G of Respiratory syncytial virus (RSV). 
     
     
         3 . The mRNA sequence according to any of  claims 1  to  2 , wherein the antigenic peptide or protein is derived from the RSV strain ATCC VR-26 long. 
     
     
         4 . The mRNA sequence according to any of  claims 1  to  3  comprising additionally
 a) a 5′-CAP structure, 
 b) a poly(A) sequence, 
 c) and optionally a poly (C) sequence. 
 
     
     
         5 . The mRNA sequence according to  claim 4 , wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides. 
     
     
         6 . The mRNA sequence according to any of  claims 1  to  5  comprising additionally at least one histone stem-loop. 
     
     
         7 . The mRNA sequence according to  claim 6 , wherein the at least one histone stem-loop is selected from following formulae (I) or (II):
 formula (I) (stem-loop sequence without stem bordering elements):   
       
         
           
           
               
               
           
         
         formula (II) (stem-loop sequence with stem bordering elements): 
       
       
         
           
           
               
               
           
         
         wherein: 
       
       
         
           
                 
                 
               
                     
                 
                   stem1 or stem2 bordering elements  
                   is a consecutive sequence of 1 to 6, preferably 
                 
                   N 1-6   
                   of 2 to 6, more preferably of 2 to 5, even more 
                 
                     
                   preferably of 3 to 5, most preferably of 4 to 5 or 
                 
                     
                   5 N, wherein each N is independently from 
                 
                     
                   another selected from a nucleotide selected from 
                 
                     
                   A, U, T, G and C, or a nucleotide analogue 
                 
                     
                   thereof; 
                 
                   stem1 [N 0-2 GN 3-5 ] 
                   is reverse complementary or partially reverse complementary 
                 
                     
                   with element stem2, and is a consecutive sequence between of 
                 
                     
                   5 to 7 nucleotides; 
                 
                     
                   wherein N 0-2  is a consecutive sequence of 0 to 2, preferably of 
                 
                     
                   0 to 1, more preferably of 1 N, wherein each N is independently 
                 
                     
                   from another selected from a nucleotide selected from A, U, T, 
                 
                     
                   G and C or a nucleotide analogue thereof; 
                 
                     
                   wherein N 3-5  is a consecutive sequence of 3 to 5, preferably of 
                 
                     
                   4 to 5, more preferably of 4 N, wherein each N is independently 
                 
                     
                   from another selected from a nucleotide selected from A, U, T, 
                 
                     
                   G and C or a nucleotide analogue thereof, and 
                 
                     
                   wherein G is guanosine or an analogue thereof, and may be 
                 
                     
                   optionally replaced by a cytidine or an analogue thereof, 
                 
                     
                   provided that its complementary nucleotide cytidine in stem2 is 
                 
                     
                   replaced by guanosine; 
                 
                   loop sequence [N 0-4 (U/T)N 0-4 ] 
                   is located between elements stem1 and stem2, and is 
                 
                     
                   a consecutive sequence of 3 to 5 nucleotides, more 
                 
                     
                   preferably of 4 nucleotides; 
                 
                     
                   wherein each N 0-4  is independent from another a 
                 
                     
                   consecutive sequence of 0 to 4, preferably of 1 to 3, 
                 
                     
                   more preferably of 1 to 2 N, wherein each N is 
                 
                     
                   independently from another selected from a 
                 
                     
                   nucleotide selected from A, U, T, G and C or a 
                 
                     
                   nucleotide analogue thereof; and 
                 
                     
                   wherein U/T represents uridine, or optionally 
                 
                     
                   thymidine; 
                 
                   stem2 [N 3-5 CN 0-2 ] 
                   is reverse complementary or partially reverse complementary 
                 
                     
                   with element stem1, and is a consecutive sequence between of 
                 
                     
                   5 to 7 nucleotides; 
                 
                     
                   wherein N 3-5  is a consecutive sequence of 3 to 5, preferably of 
                 
                     
                   4 to 5, more preferably of 4 N, wherein each N is independently 
                 
                     
                   from another selected from a nucleotide selected from A, U, T, 
                 
                     
                   G and C or a nucleotide analogue thereof; 
                 
                     
                   wherein N 0-2  is a consecutive sequence of 0 to 2, preferably of 
                 
                     
                   0 to 1, more preferably of 1 N, wherein each N is independently 
                 
                     
                   from another selected from a nucleotide selected from A, U, T, 
                 
                     
                   G and C or a nucleotide analogue thereof; and 
                 
                     
                   wherein C is cytidine or an analogue thereof, and may be 
                 
                     
                   optionally replaced by a guanosine or an analogue thereof 
                 
                     
                   provided that its complementary nucleotide guanosine in stem1 
                 
                     
                   is replaced by cytidine; 
                 
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein 
         stem1 and stem2 are capable of base pairing with each other 
         forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or 
         forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2. 
       
     
     
         8 . The mRNA sequence according to  claim 7 , wherein the at least one histone stem-loop is selected from at least one of following formulae (Ia) or (IIa): 
       
         
           
           
               
               
           
         
         formula (Ia) (stem-loop sequence without stem bordering elements) 
       
       
         
           
           
               
               
           
         
         formula (IIa) (stem-loop sequence with stem bordering elements) 
       
     
     
         9 . The mRNA sequence according to any of  claims 1  to  8  comprising additionally a 3′-UTR element. 
     
     
         10 . The mRNA sequence according to  claim 9 , wherein the at least one 3′UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′UTR of a gene providing a stable mRNA or from a homolog, a fragment or a variant thereof. 
     
     
         11 . The mRNA sequence according to  claim 10 , wherein the 3′UTR element comprises or consists of a nucleic acid sequence derived from a 3′UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof. 
     
     
         12 . The mRNA sequence according to  claim 11 , wherein the 3′-UTR element comprises or consists of a nucleic acid sequence derived from a 3′UTR of α-globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 29, a homolog, a fragment, or a variant thereof; 
     
     
         13 . The mRNA sequence according to any of  claims 9  to  12 ; wherein the mRNA sequence comprises, preferably in 5′- to 3′-direction:
 a.) a 5′-CAP structure, preferably m7GpppN; 
 b.) a coding region encoding at least one antigenic peptide or protein of Respiratory syncytial virus (RSV), preferably derived from the fusion protein F of Respiratory syncytial virus (RSV); 
 c.) a 3′-UTR element comprising or consisting of a nucleic acid sequence which is derived from a alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 29, a homolog, a fragment or a variant thereof; 
 d.) a poly(A) sequence, preferably comprising 64 adenosines; 
 e.) a poly(C) sequence, preferably comprising 30 cytosines; and 
 f.) a histone-stem-loop, preferably comprising the corresponding RNA sequence to the nucleic acid sequence according to SEQ ID No 30. 
 
     
     
         14 . The mRNA sequence according to  claim 13 , wherein the mRNA sequence comprises the RNA sequence according to SEQ ID No. 31. 
     
     
         15 . The mRNA sequence according to  claim 9 , wherein the at least one 3′UTR element comprises or consist of a nucleic acid sequence which is derived from the 3′UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3′UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3′UTR of a human albumin gene or from a variant thereof, even more preferably from the 3′UTR of the human albumin gene according to GenBank Accession number NM_000477.5, or from a fragment or variant thereof. 
     
     
         16 . The mRNA sequence according to  claim 15 , wherein the 3′UTR element is derived from a nucleic acid sequence according to SEQ ID NO. 25, preferably from a corresponding RNA sequence, a homolog, a fragment or a variant thereof. 
     
     
         17 . The mRNA sequence according to any of  claims 1  to  16  comprising additionally a 5′-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5′UTR of a TOP gene preferably from a corresponding RNA sequence, a homolog, a fragment, or a variant thereof, preferably lacking the 5′TOP motif. 
     
     
         18 . The mRNA sequence according to  claim 17 , wherein the 5′UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′UTR of a TOP gene encoding a ribosomal protein, preferably from a corresponding RNA sequence or from a homolog, a fragment or a variant thereof, preferably lacking the 5′TOP motif. 
     
     
         19 . The mRNA sequence according to  claim 18 , wherein the 5′UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a homolog, a fragment or variant thereof, preferably lacking the 5′TOP motif and more preferably comprising or consisting of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 23. 
     
     
         20 . The mRNA sequence according to  claim 19 ; wherein the mRNA sequence comprises, preferably in 5′- to 3′-direction:
 a.) a 5′-CAP structure, preferably m7GpppN; 
 b.) a 5′-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a TOP gene, preferably comprising or consisting of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 23, a homolog, a fragment or a variant thereof; 
 c.) a coding region encoding at least one antigenic peptide or protein of Respiratory syncytial virus (RSV), preferably derived from the fusion protein F of Respiratory syncytial virus (RSV); 
 d.) a 3′UTR element comprising or consisting of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO. 18, a homolog, a fragment or a variant thereof; 
 e.) a poly(A) sequence preferably comprising 64 adenosines; 
 f.) a poly(C) sequence, preferably comprising 30 cytosines; and 
 g.) a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID No 30. 
 
     
     
         21 . The mRNA sequence according to  claim 20 , wherein the mRNA sequence comprises the RNA sequence according to SEQ ID No. 32 or 33. 
     
     
         22 . The mRNA sequence according to  claims 1  to  21 , wherein the mRNA sequence is associated with or complexed with a cationic or polycationic compound or a polymeric carrier, optionally in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNA to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9. 
     
     
         23 . The mRNA sequence according to  claim 22 , wherein the mRNA sequence is associated or complexed with a cationic protein or peptide, preferably protamine. 
     
     
         24 . A composition comprising a plurality or more than one of mRNA sequences each according to any of  claims 1  to  23 . 
     
     
         25 . Pharmaceutical composition comprising an mRNA sequence as defined according to any of  claims 1  to  23  or a composition as defined according to  claim 24  and optionally a pharmaceutically acceptable carrier. 
     
     
         26 . Pharmaceutical composition according to  claim 25 , wherein the mRNA sequence is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides and most preferably protamine. 
     
     
         27 . Pharmaceutical composition according to  claim 26 , wherein the ratio of complexed mRNA to free mRNA is selected from a range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexed mRNA to free mRNA is selected from a ratio of 1:1 (w/w). 
     
     
         28 . Kit or kit of parts comprising the components of the mRNA sequence as defined according to any of  claims 1  to  23 , the composition as defined according to  claim 24 , the pharmaceutical composition as defined according to any of  claims 25  to  27  and optionally technical instructions with information on the administration and dosage of the components. 
     
     
         29 . mRNA sequence as defined according to any of  claims 1  to  23 , composition as defined according to  claim 24 , pharmaceutical composition as defined according to any of  claims 25  to  27 , and kit or kit of parts as defined according to  claim 28  for use as a medicament. 
     
     
         30 . mRNA sequence as defined according to any of  claims 1  to  23 , composition as defined according to  claim 24 , pharmaceutical composition as defined according to any of  claims 25  to  27 , and kit or kit of parts as defined according to  claim 28  for use in the treatment or prophylaxis of RSV infections. 
     
     
         31 . mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to  claim 30 , wherein the treatment is combined with administration of RSV immune globuline, particularly Palivizumab. 
     
     
         32 . A method of treatment or prophylaxis of RSV infections comprising the steps:
 a) providing the mRNA sequence as defined according to any of  claims 1  to  23 , the composition as defined according to  claim 24 , the pharmaceutical composition as defined according to any of  claims 25  to  27 , or the kit or kit of parts as defined according to  claim 28 ;   b) applying or administering the mRNA sequence, the composition, the pharmaceutical composition or the kit or kit of parts to a tissue or an organism;   c) optionally administering RSV immune globuline.